The Expression of STAT3 and STAT5A Genes in Severe Refractory Asthma.

BACKGROUND
Despite being a high burden disorder, the pathogenesis of severe refractory asthma (SRA) is poorly understood. There are some evidences for the involvement of members of the signal transducer and activator of transcription (STAT) family, including STAT3 and STAT5a. Our study aimed to evaluate the gene expression of STAT3 and STAT5a in asthma and SRA to establish if there is an association.


MATERIALS AND METHODS
Using quantitative real-time polymerase chain reactions (qRT-PCR), the transcript levels of STAT3 and STAT5a were evaluated in peripheral blood mononuclear lymphocytes (PBML) isolated from 13 patients with SRA, 14 with mild asthma, and 30 healthy volunteers.


RESULTS
There were no significant differences in STAT3 transcript levels between study groups. There was however a significant difference in STAT5a transcript levels between cases and controls (p-value=0.03). In comparison to healthy controls, the levels of STAT5a were notably lower in patients with mild asthma and significantly least in those with SRA.


CONCLUSION
Our study found no appreciable association between STAT3 gene expression and either mild asthma or SRA. However, the STAT5a down regulation in asthmatics and especially SRA is a notable finding which denotes on association between STAT5a and different level of asthma.


INTRODUCTION
Chronic asthma is a non-communicable inflammatory airway disorder in which patients present with recurring bouts of breathlessness and wheezing. Although the exact pathogenesis of asthma is not fully understood, numerous causative environmental and/or triggering agents have been described. These include allergens, tobacco smoke, chemical irritants, and microorganisms. These factors interact with an individual's genetic and epigenetic background, leading to the development of asthma or the triggering of attacks (1).
Asthma is suspected to cause approximately 250,000 premature deaths annually and the World Health Organization (WHO) estimates that more than 300 million individuals are affected worldwide. This number is predicted to increase to 400 million by 2025. Due to the fact that asthma is a major cause of disability, poor quality of life, increased health resource utilization, and is a public health concern, it is essential to fully understand the genetic basis of the disease (2,3).
During previous decades, there has been some controversy concerning the definition and classification of severe refractory asthma (SRA), found in approximately in 5-10% of cases (4). In 2011, an international consensus statement was published by the Innovative Medicine Initiative (IMI) that aimed to clarify a unique definition, classification, and diagnostic algorithm for SRA. This stated that "the term 'severe refractory asthma' should be TANAFFOS reserved for patients with asthma in whom alternative diagnoses have been excluded, comorbidities have been treated, trigger factors have been removed (if possible) and compliance with treatment has been checked, but still have poor asthma control or frequent (≥2) severe exacerbations per year despite the prescription of high-intensity treatment or can only maintain adequate control when taking systemic corticosteroids and are thereby at risk of serious adverse effects of treatment" (2).
In order to understand the fundamental mechanisms of asthma pathogenesis, efforts have been made to identify genetic associations with asthma and SRA (5,6). Among the numerous genes known to associate with asthma, members of the signal transducer and activator of transcription (STAT) pathway appear to have an important function (7).
Seven proteins of this family have been shown to have roles in signal transduction pathways and/or gene transcription (8) and may be involved with the mechanisms that underlie asthma and SRA.
The gene coding for STAT3 is located in chromosomal region 17q21.2 and consists of 24 exons. Through alternative splicing, STAT3 can be expressed as three different splice variants. STAT3 is an essential protein involved in cell growth and apoptosis, and is expressed in all tissue types. Furthermore, it can act as a transcription factor and may also be a co-activator of signal transduction by glucocorticoid receptors (9,10).
Like STAT3, STAT5 is related to glucocorticoid receptors and acts as a transcription activator in the immune system (11). There are two isoforms of STAT5 (STAT5a and STAT5b), coded by two separate genes located at inverted positions within the 17q21.2 or 17q11.2 genomic regions (12,13). Several important cellular processes are influenced by STAT5 isoforms, including replication, apoptosis, differentiation, and inflammation. It has also been shown that STAT5a/b are important for lymphocyte proliferation, apoptosis, and have been used for targeted gene therapy and therapeutics (e.g., for asthma and cancer) (14)(15)(16)(17).
There is some evidence supporting the involvement STAT3 and STAT5 in the development of asthma (18,19).
STAT3 has been demonstrated to be involved in airway inflammation, allergy, and asthma through several proposed mechanisms. These include the Th2/Th17 immune responses and epidermal growth factor receptor (EGFR) signaling (20)(21)(22). Furthermore, it has been proposed that STAT3 is a potential target for asthma and SRA therapeutics (23). However, there are also several studies that discount a role for STAT3 in asthma (24,25). This suggests that STAT5 may be involved in asthma through mast cell pathogenesis (8,26) and there have been several studies supporting such a link (27)(28)(29)(30)(31). The association between asthma and the STAT5b isoform has been the most well studied relationship to date but a potential role for the STAT5a isoform is unclear (14). A previous study by Tsitsiou et al. that evaluated gene expression in patients with severe asthma reported that STAT3 and STAT5b expression levels were 1.59 and 1.62 times higher, respectively in these patients (3). Our study, therefore, aimed to investigate STAT3 and STAT5 gene expression in asthma and SRA.     Finally, the ethnicities of participants were found to be similar in each of the three groups (60% Fars, 8% Turks, 12% Lores, and 20% Kurds).

Comparison of Transcript Abundance
Student's t-tests comparing the qRT-PCR data revealed that the transcript expressions of STAT3 and STAT5A were not significantly different between genders (Tables 3 and   4). STAT3 and STAT5a transcript expressions also did not correlate with age of all participants (r=0.42, P-value=0.35 for STAT3 and r=0.31, P-value=0.67 for STAT5a).

Furthermore the gene expression (mean ΔCts) of STAT3
and STAT5A were compared by ANOVA statistical tests between cases and control groups. showed the expression ratios of asthma and SRA groups against the control group are 1.096 and 1.013 respectively, when the control adjusted to 1 (Figure 1). These findings show the level of STAT3 gene expression in asthma group is followed by SRA and control groups.
When examining STAT5a transcript levels, we found a significant difference between the disease groups (P-value=0.03) ( Table 4). A Tukey's post hoc test suggested that the only significant difference was between healthy and SRA groups (P-value=0.04). The P-value for a putative difference between the mild asthma versus SRA groups, and the asthma versus healthy groups, were 0.83 and 0.15, respectively. Fold change analysis (2 -ΔΔCt ) revealed the highest STAT5a transcript levels were in healthy controls, followed by the mild asthma and then SRA groups (the control group was set to 1.0) (Figure 2).  As the steroid regimens and pulmonary function tests of all participants were variable, there was possibility that these factors interfere with our results. To reduce these effects, we applied certain standards restrictedly to prevent bias. For example, the cases were selected if they had diagnostic criteria of GINA (for asthma) and IMI (for SRA) for more than 2 years and had no experience of exacerbation in the 6 months prior to sampling.
Furthermore, they were controlled by inhaled corticosteroids which have least systemic effects (34).

STAT3
As previously mentioned, the role of STAT3 in the pathogenesis of asthma and SRA is somewhat controversial. Our study revealed that STAT3 gene expression was not significantly different between the three groups of healthy controls, asthma and severe asthma. This finding is compatible with some reports. For example, Chiba et al. found that STAT3 had no notable role in the pathogenesis of bronchial allergic asthma (24,25).
Based on our results and previous studies, we hypothesize that, STAT3 may have some role in the metabolic pathways of asthma, however, it does not seem to be directly involved in the pathogenesis of asthma and SRA. Meanwhile, the controversies in studies may be due to different methods used in each study, the individual role of the three STAT3 isoforms, an inadequate sample size, or even unknown confounding factors.

STAT5a
In contrast to STAT3, we found a significant difference in the transcript levels of STAT5a between the study groups. The STAT5a expression in healthy controls was nearly twice of patients with asthma. The groups of patients with SRA had even less transcript, approximately 20% lower than patients with mild asthma. This demonstrates that there is STAT5a down regulation in asthma and SRA cases, suggesting that this isoform has a role in the pathogenesis of asthma, particularly its severe form.
The genome wide association studies (GWAS) have found that severe asthma is associated with the 17q21 chromosomal region; the region where codes some proteins like STAT5a (6). Furthermore, many evidences signified the role of STAT5a in asthma (18,27), such as that by Stefanowicz et al, who found that STAT5a gene expression is decreased in the epithelial cells of airways (41).
Although the exact mechanisms of asthma pathogenesis remain unclear, some studies have suggested possible mechanisms for how STAT5a may be involved.
These include roles for STAT5a in controlling IL-9 expression, the differentiation of Th2, Th9, and Th17 cells (19), the activity of the CD69 receptor and its regulatory role in Th17 cells (42), several mast cell pathways (8,26), the activity of glucocorticoid receptors (11)

CONCLUSION
In conclusion, we found no evidence to support the suggestion that STAT3 is involved in asthma and SRA.
Further investigation may provide more information to elucidate its role in respiratory inflammation disorders.
Meanwhile, down regulation of STAT5a in asthma, and especially SRA, is a notable finding which worth to be considered more in future studies. Using transcriptomic and proteomic methods with higher sample size, the expression study of STAT5a, STAT5b and total STAT5 may provide considerable results on their roles in asthma pathogenesis.